Culture techniques for ciliate protozoa from the rumen: Recent advances and persistent challenges.

Ciliate protozoa are key members of the microbial community of the rumen. Their study is important to the health and productivity of cattle, which are their hosts. However, there have been persistent challenges in culturing this microbial group in the laboratory. This review will sum up recent advances along with these persistent challenges. Protozoa have been maintained in three types of cultures (ex vivo, in vitro batch, in vitro continuous). Ex vivo cultures are prepared readily from rumen contents by washing away contaminating cells (e.g., bacteria). They have been useful in making basic observations of metabolism, such as which types of fermentation products protozoa form. However, these cultures can be maintained for only short periods (minutes or hours). In vitro batch and in vitro continuous cultures can be used in longer experiments (weeks or longer). However, it is not currently possible to maintain protozoa in these cultures unless bacteria are also present. We conclude the review with a protocol for preparing ex vivo cultures of protozoa. Our protocol has been standardized and used successfully across animal diets, users, and institutions. We anticipate this review will prepare others to culture rumen ciliate protozoa and reach new insights into this important microbial group.

Energy conservation involving 2 respiratory circuits.

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.

Using neural networks to mine text and predict metabolic traits for thousands of microbes.

Microbes can metabolize more chemical compounds than any other group of organisms. As a result, their metabolism is of interest to investigators across biology. Despite the interest, information on metabolism of specific microbes is hard to access. Information is buried in text of books and journals, and investigators have no easy way to extract it out. Here we investigate if neural networks can extract out this information and predict metabolic traits. For proof of concept, we predicted two traits: whether microbes carry one type of metabolism (fermentation) or produce one metabolite (acetate). We collected written descriptions of 7,021 species of bacteria and archaea from Bergey's Manual. We read the descriptions and manually identified (labeled) which species were fermentative or produced acetate. We then trained neural networks to predict these labels. In total, we identified 2,364 species as fermentative, and 1,009 species as also producing acetate. Neural networks could predict which species were fermentative with 97.3% accuracy. Accuracy was even higher (98.6%) when predicting species also producing acetate. Phylogenetic trees of species and their traits confirmed that predictions were accurate. Our approach with neural networks can extract information efficiently and accurately. It paves the way for putting more metabolic traits into databases, providing easy access of information to investigators.

Accurate estimation of microbial sequence diversity with Distanced.

MOTIVATION: Microbes are the most diverse organisms on the planet. Deep sequencing of ribosomal DNA (rDNA) suggests thousands of different microbes may be present in a single sample. However, errors in sequencing have made any estimate of within-sample (alpha) diversity uncertain. RESULTS: We developed a tool to estimate alpha diversity of rDNA sequences from microbes (and other sequences). Our tool, Distanced, calculates how different (distant) sequences would be without sequencing errors. It does this using a Bayesian approach. Using this approach, Distanced accurately estimated alpha diversity of rDNA sequences from bacteria and fungi. It had lower root mean square prediction error (RMSPE) than when using no tool (leaving sequencing errors uncorrected). It was also accurate with non-microbial sequences (antibody mRNA). State-of-the-art tools (DADA2 and Deblur) were far less accurate. They often had higher RMSPE than when using no tool. Distanced thus represents an improvement over existing tools. Distanced will be useful to several disciplines, given microbial diversity affects everything from human health to ecosystem function. AVAILABILITY AND IMPLEMENTATION: Distanced is freely available at https://github.com/thackmann/Distanced. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A New Pathway for Forming Acetate and Synthesizing ATP during Fermentation in Bacteria.

Many bacteria and other organisms carry out fermentations forming acetate. These fermentations have broad importance for foods, agriculture, and industry. They also are important for bacteria themselves because they often generate ATP. Here, we found a biochemical pathway for forming acetate and synthesizing ATP that was unknown in fermentative bacteria. We found that the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. It did not use phosphotransacetylase or acetate kinase, enzymes found in nearly all acetate-forming bacteria. Instead, it used a pathway involving two different enzymes. The first enzyme, succinyl coenzyme A (succinyl-CoA):acetate CoA-transferase (SCACT), forms acetate from acetyl-CoA. The second enzyme, succinyl-CoA synthetase (SCS), synthesizes ATP. We identified the genes encoding these enzymes, and they were homologs of SCACT and SCS genes found in other bacteria. The pathway resembles one described in eukaryotes, but it uses bacterial, not eukaryotic, gene homologs. To find other instances of the pathway, we analyzed sequences of all biochemically characterized homologs of SCACT and SCS (103 enzymes from 64 publications). Homologs with similar enzymatic activity had similar sequences, enabling a large-scale search for them in genomes. We searched nearly 600 genomes of bacteria known to form acetate, and we found that 6% encoded homologs with SCACT and SCS activity. This included >30 species belonging to 5 different phyla, showing that a diverse range of bacteria encode the SCACT/SCS pathway. This work suggests the SCACT/SCS pathway is important for acetate formation in many branches of the tree of life. IMPORTANCE Pathways for forming acetate during fermentation have been studied for over 80 years. In that time, several pathways in a range of organisms, from bacteria to animals, have been described. However, one pathway (involving succinyl-CoA:acetate CoA-transferase and succinyl-CoA synthetase) has not been reported in prokaryotes. Here, we discovered enzymes for this pathway in the fermentative bacterium Cutibacterium granulosum. We also found >30 other fermentative bacteria that encode this pathway, demonstrating that it could be common. This pathway represents a new way for bacteria to form acetate from acetyl-CoA and synthesize ATP via substrate-level phosphorylation. It could be a target for controlling yield of acetate during fermentation, with relevance for foods, agriculture, and industry.

Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

Genomic diversification of giant enteric symbionts reflects host dietary lifestyles.

Herbivorous surgeonfishes are an ecologically successful group of reef fish that rely on marine algae as their principal food source. Here, we elucidated the significance of giant enteric symbionts colonizing these fishes regarding their roles in the digestive processes of hosts feeding predominantly on polysiphonous red algae and brown Turbinaria algae, which contain different polysaccharide constituents. Using metagenomics, single-cell genomics, and metatranscriptomic analyses, we provide evidence of metabolic diversification of enteric microbiota involved in the degradation of algal biomass in these fishes. The enteric microbiota is also phylogenetically and functionally simple relative to the complex lignocellulose-degrading microbiota of terrestrial herbivores. Over 90% of the enzymes for deconstructing algal polysaccharides emanate from members of a single bacterial lineage, "Candidatus Epulopiscium" and related giant bacteria. These symbionts lack cellulases but encode a distinctive and lineage-specific array of mostly intracellular carbohydrases concurrent with the unique and tractable dietary resources of their hosts. Importantly, enzymes initiating the breakdown of the abundant and complex algal polysaccharides also originate from these symbionts. These are also highly transcribed and peak according to the diel lifestyle of their host, further supporting their importance and host-symbiont cospeciation. Because of their distinctive genomic blueprint, we propose the classification of these giant bacteria into three candidate genera. Collectively, our findings show that the acquisition of metabolically distinct "Epulopiscium" symbionts in hosts feeding on compositionally varied algal diets is a key niche-partitioning driver in the nutritional ecology of herbivorous surgeonfishes.

Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose.

Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.IMPORTANCE We introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short-chain fatty acids.

Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here, we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% of bacteria had an atypical pathway for propionate or succinate formation; 6% of bacteria had an atypical pathway for butyrate formation and 33% of bacteria had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways - a common goal of omics studies - could be incorrect if well-recognized pathways are used for reference. Furthermore, it calls for renewed efforts to delineate fermentation pathways biochemically.

Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli.

Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [(14)C]glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the Vmax of 2-NBDG transport was 2.9-fold lower than that for [(14)C]glucose, and the Km was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [(14)C]mannose and [(14)C]deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement.

Targeted disruption of CD1d prevents NKT cell development in pigs.

Studies in mice genetically lacking natural killer T (NKT) cells show that these lymphocytes make important contributions to both innate and adaptive immune responses. However, the usefulness of murine models to study human NKT cells is limited by the many differences between mice and humans, including that their NKT cell frequencies, subsets, and distribution are dissimilar. A more suitable model may be swine that share many metabolic, physiological, and growth characteristics with humans and are also similar for NKT cells. Thus, we analyzed genetically modified pigs made deficient for CD1d that is required for the development of Type I invariant NKT (iNKT) cells that express a semi-invariant T-cell receptor (TCR) and Type II NKT cells that use variable TCRs. Peripheral blood analyzed by flow cytometry and interferon-γ enzyme-linked immuno spot assays demonstrated that CD1d-knockout pigs completely lack iNKT cells, while other leukocyte populations remain intact. CD1d and NKT cells have been shown to be involved in shaping the composition of the commensal microbiota in mice. Therefore, we also compared the fecal microbiota profile between pigs expressing and lacking NKT cells. However, no differences were found between pigs lacking or expressing CD1d. Our results are the first to show that knocking-out CD1d prevents the development of NKT cells in a non-rodent species. CD1d-deficient pigs should offer a useful model to more accurately determine the contribution of NKT cells for human immune responses. They also have potential for understanding how NKT cells impact the health of commercial swine.

Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.

The vast landscape of carbohydrate fermentation in prokaryotes.

Fermentation is a type of metabolism carried out by organisms in environments without oxygen. Despite being studied for over 185 years, the diversity and complexity of this metabolism are just now becoming clear. Our review starts with the definition of fermentation, which has evolved over the years and which we help further refine. We then examine the range of organisms that carry out fermentation and their traits. Over one-fourth of all prokaryotes are fermentative, use more than 40 substrates, and release more than 50 metabolic end products. These insights come from studies analyzing records of thousands of organisms. Next, our review examines the complexity of fermentation at the biochemical level. We map out pathways of glucose fermentation in unprecedented detail, covering over 120 biochemical reactions. We also review recent studies coupling genomics and enzymology to reveal new pathways and enzymes. Our review concludes with practical applications for agriculture, human health, and industry. All these areas depend on fermentation and could be improved through manipulating fermentative microbes and enzymes. We discuss potential approaches for manipulation, including genetic engineering, electrofermentation, probiotics, and enzyme inhibitors. We hope our review underscores the importance of fermentation research and stimulates the next 185 years of study.

New biochemical pathways for forming short-chain fatty acids during fermentation in rumen bacteria.

Short-chain fatty acids (SCFA) are essential to cattle as a source of energy and for other roles in metabolism. These molecules are formed during fermentation by microbes in the rumen, but even after decades of study, the biochemical pathways responsible for forming them are not always clear. Here we review recent advances in this area and their importance for improving animal productivity. Studies of bacterial genomes have pointed to unusual biochemical pathways in rumen organisms. One study found that 8% of rumen organisms forming acetate, a major SCFA, had genes for a pathway previously unknown in bacteria. The existence of this pathway was subsequently confirmed biochemically in propionibacteria. The pathway was shown to involve 2 enzymes that convert acetyl-coenzyme A to acetate. Similar studies have revealed new enzymatic steps for forming propionate and butyrate, other major SCFA. These new steps and pathways are significant for controlling fermentation. With more precise control over SCFA, cows can be fed more precisely and potentially reach higher productivity.

Quantifying the responses of mixed rumen microbes to excess carbohydrate.

The aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P = 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P < 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO2, CH4, and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.

The phenotype and genotype of fermentative prokaryotes.

Fermentation is a type of metabolism pervasive in oxygen-deprived environments. Despite its importance, we know little about the range and traits of organisms that carry out this metabolism. Our study addresses this gap with a comprehensive analysis of the phenotype and genotype of fermentative prokaryotes. We assembled a dataset with phenotypic records of 8350 organisms plus 4355 genomes and 13.6 million genes. Our analysis reveals fermentation is both widespread (in ~30% of prokaryotes) and complex (forming ~300 combinations of metabolites). Furthermore, it points to previously uncharacterized proteins involved in this metabolism. Previous studies suggest that metabolic pathways for fermentation are well understood, but metabolic models built in our study show gaps in our knowledge. This study demonstrates the complexity of fermentation while showing that there is still much to learn about this metabolism. All resources in our study can be explored by the scientific community with an online, interactive tool.

The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella.

Propionate is a microbial metabolite formed in the gastrointestinal tract, and it affects host physiology as a source of energy and signaling molecule. Despite the importance of propionate, the biochemical pathways responsible for its formation are not clear in all microbes. For the succinate pathway used during fermentation, a key enzyme appears to be missing-one that oxidizes ferredoxin and reduces NAD. Here we show that Rnf [ferredoxin-NAD+ oxidoreductase (Na+-transporting)] is this key enzyme in two abundant bacteria of the rumen (Prevotella brevis and Prevotella ruminicola). We found these bacteria form propionate, succinate, and acetate with the classic succinate pathway. Without ferredoxin:NAD+ oxidoreductase, redox cofactors would be unbalanced; it would produce almost equal excess amounts of reduced ferredoxin and oxidized NAD. By combining growth experiments, genomics, proteomics, and enzyme assays, we point to the possibility that these bacteria solve this problem by oxidizing ferredoxin and reducing NAD with Rnf [ferredoxin-NAD+ oxidoreductase (Na+-transporting)]. Genomic and phenotypic data suggest many bacteria may use Rnf similarly. This work shows the ferredoxin:NAD+ oxidoreductase activity of Rnf is important to propionate formation in Prevotella species and other bacteria from the environment, and it provides fundamental knowledge for manipulating fermentative propionate production.

Redefining the coenzyme A transferase superfamily with a large set of manually annotated proteins.

The coenzyme A (CoA) transferases are a superfamily of proteins central to the metabolism of acetyl-CoA and other CoA thioesters. They are diverse group, catalyzing over a 100 biochemical reactions and spanning all three domains of life. A deeply rooted idea, proposed two decades ago, is these enzymes fall into three families (I, II, and III). Here we find they fall into different families, which we achieve by analyzing all CoA transferases characterized to date. We manually annotated 94 CoA transferases with functional information (including rates of catalysis for 208 reactions) from 97 publications. This represents all enzymes we could find in the primary literature, and it is double the number annotated in four protein databases (BRENDA, KEGG, MetaCyc, UniProt). We found family I transferases are not closely related to each other in terms of sequence, structure, and reactions catalyzed. This family is not even monophyletic. These problems are solved by regrouping the three families into six, including one family with many non-CoA transferases. The problem (and solution) became apparent only by analyzing our large set of manually annotated proteins. It would have been missed if we had used the small number of proteins annotated in UniProt and other databases. Our work is important to understanding the biology of CoA transferases. It also warns investigators doing phylogenetic analyses of proteins to go beyond information in databases.

A system for predicting energy and protein requirements of wild ruminants.

Wild ruminants require energy and protein for the normal function. I developed a system for predicting these energy and protein requirements across ruminant species and life stages. This system defines requirements on the basis of net energy (NE), net protein (NP), and ruminally degraded protein (RDP). Total NE and NP requirements are calculated as the sum of NE and NP required for several functions (maintenance, activity, thermoregulation, gain, lactation, and gestation). To estimate the requirements for each function, I collected data predominantly for wild species and then formulated allometric and other equations that predict requirements across species. I estimated RDP requirements using an equation for cattle. I then related NE, NP, and RDP to quantities more practical for diet formulation (e.g. dry matter intake). I tabulated requirements over a range of body mass and life stages (neonate, juvenile, nonproductive adult, lactating adult, and gestating adult). Tabulated requirements suggest that adults at peak lactation require greatest quantities of energy and neonates generally require greatest quantities of protein, agreeing with suggestions that lactation is energetically expensive and protein is most limiting during growth. Equations used in this system were precise (allometric equations had R(2) generally ≥0.89 and coefficient of variation <31.1%) and expected to reliably predict requirements across species. Results showed that a system for beef cattle would overestimate NE and either over- or underestimate NP for gain when applied to wild ruminants, showing that systems for wild ruminants should not extrapolate from requirements for domestic ruminants. One prominent system for wild ruminants predicted at times vastly different protein requirements from those predicted by the proposed system. The proposed system should be further evaluated and expanded to include other nutrients.